Materials

TRIzol reagent was obtained from Ambion-Applied Biosystems (Austin, TX). Conventional polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) reagents were purchased from Clontech Laboratories (Mountain View, CA) and Applied Biosystems (Carlsbad, CA). Human USM cells and culture media were purchased from Lonza (Wakersville, MD). C57-Bl6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). All other pharmaceutical drug reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise indicated.

Cell culture

MUSM cells were harvested for use in culture using an enzymatic cellular dissociation kit (Worthington, Lakewood, NJ) and grown in SmBMII media with the manufacturer’s recommended additives (SmGM-2 Singlequots Kit supplemntal and growth factors; Lonza, Walkersville, MD). Nonpregnant human USM cells were purchased from Lonza (CC-2562) and grown in SMBMII as above. Cells were grown to confluence in Tm75 cm ² flasks in a humidified 37°C incubator in 95% air/5% CO ₂ .

RNA extraction and reverse transcription of cDNA in murine and human USM

Total RNA was extracted from primary MUSM cells, flash frozen whole mouse brain (positive control), and cultured human USM cells using Trizol according to the manufacturer’s recommendations. Human brain total RNA was purchased from Clontech Laboratories (#636530). RNA purity and quantity was measured by 260/280 nM absorbance (Beckman Du 640; Beckman Coulter, Inc., Pasadena, CA). Total RNA from human and murine USM was diluted in RNAse free water to a final concentration 500 μg/mL, aliquoted and stored at −80°C. Complementary DNA synthesis was performed (1 μg total RNA in a final volume of 20 μL) with MMLV RT Reverse Transcriptase-Advantage for PCR (Clontech Laboratories) using either oligo (dT) or random hexamer priming following the manufacturer’s recommendations.

Reverse transcription polymerase chain reaction

Newly synthesized cDNA (5 μL of original 20 μL reaction) was used in PCR with the Advantage 2 PCR Kit on an MJ Research PTC-200 Peltier thermal cycler (Bio-Rad, Hercules, CA) following the manufacturer’s protocol. Sense and antisense primers specific for ANO1 and 2 were constructed using primer blast ( Table ) and were purchased from Invitrogen (Carlsbad, CA). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Primers were designed so that amplicon size for mRNA and genomic DNA were easily distinguishable. cDNA samples were initially denatured at 94°C for 30 seconds. The annealing temperatures for ANO1 and 2 were set to 64°C and 68°C, respectively, based on previously obtained optimal temperatures. For RT-PCR, there was a completion step of 30 seconds. PCR products were analyzed on 10% polyacrylamide gels stained with ethidium bromide (Molecular Probes, Eugene, OR) and visualized using a gel imager and VisionWorks software (Biospectra UVP, Upland, CA).

Immunohistochemistry of MUSM for ANO1 and 2 expression

Uterine smooth muscle was dissected from nonpregnant C57-bl6 mice (18-20 g) and immediately fixed in 4% paraformaldehyde (4 ^° C overnight), then incubated in 30% sucrose in PBS for an additional 24 hours before processing for cryostat sectioning. The sections (6 μm) were washed in PBS, incubated with 0.1% Triton X-100 for 10 minutes, blocked with 15% goat serum, then incubated overnight at 4 ^° C in primary antisera. To confirm that protein expression persists in primary culture conditions, parallel experiments were conducted using primary murine USM cells. These cells were cultured on coverslips in SmBMII smooth muscle media, containing 5% FBS and other manufacturer recommended additives, for 2-3 days then fixed with 3% paraformaldehyde in PBS for 5 minutes. Primary antibodies used were (1) anti-ANO1 (rabbit, monoclonal; Abcam #ab64085, 1:100 dilution in PBS) for cultured cell immunocytochemistry, (2) anti-ANO1 (rabbit polyclonal; Abcam #ab53213, 1:1 dilution in PBS; Abcam, Cambridge, MA) for murine uterine tissue immunohistochemistry, and (3) Rhodamine Phalloidin for cultured cell actin cytoskeleton immunocytochemistry (1:100; Life Technologies, Grand Island, NY).

Secondary antibodies consisted of fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:400 dilution), incubated for 1 hour. Nuclear staining was achieved using mounting medium premixed with 4′,6-diamidino-2-phenylindole (DAPI) stain (H-1500; Vector Laboratories, Burlingame, CA). Negative controls were performed on serial sections by omitting primary antibody. All the immunofluorescence experiments were repeated at least 3 times. Samples were viewed under confocal microscopy (Nikon Eclipse; Nikon, Tokyo, Japan) and images were acquired with NIS software version 4.10 (Nikon).

Immunoblot for ANO1/2 protein expression

Mouse uterine primary cultured and human airway smooth muscle cells (positive control) were used for Western blot following homogenization in ice-cold NP40 cell lysis buffer (50 mM Tris, 250 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 50 mM NaF 1 mM Na3VO4, 1% nonide P40, and 0.02% NaN ₃ ). Following centrifugation (5000 g, 5 minutes, 4°C) of the whole cell lysate, the supernatants were saved and protein concentrations were determined. Aliquots were solubilized by heating at 95°C for 5 minutes in sample buffer, were subsequently electrophoresed through a 4-15% Mini-PROTEAN TGX gel (Bio-Rad), transferred to polyvinylidene fluoride, and probed with TMEM16A antibody (rabbit polyclonal was diluted to 1:4, no. ab53213; Abcam). Equivalent loading was confirmed by commensurate probing with β-actin (rabbit monoclonal diluted to 1:1,000; Millipore, Billerica, MA). The primary antibodies were detected by horseradish peroxidase-conjugated goat antirabbit antibodies (1:3000 for TMEM16A; 1:5000 for β-actin). The signal from the immunoreactive bands was detected by enhanced chemiluminescence (SuperSignal West Femto no. 34095; Thermo Scientific, Rockford, IL) according to the manufacturer’s recommendation. Digital acquisition of the resulting bands was achieved using UVP biochemi system (UVP, Llc, Upland, CA) and Vision Works LS software (VisionWorks, Inc., Cedar Rapids, IA).

Patch clamp electrophysiologic studies in MUSM cells

To assess membrane potential currents relevant to the physiology of contractility, we performed whole cell patch clamp studies. MUSM was digested with collagenase type IV at 37°C for 5-10 minutes. The released cells were placed on poly10 L-Lysine 12 mm coverslips (BD, San Jose, CA), coated with 0.5 mg/mL collagen Type I (Sigma, St. Louis, MO) and incubated for another 1-3 days. For whole cell recordings, coverslips were transferred to a 0.5 mL chamber on the stage of an inverted microscope (Nikon). Membrane currents were recorded using whole-cell configuration. The extracellular solution contained (in mM) 130.0 NaCl, 5.5 tetraethylammonium chloride, 2.2 CaCl ₂ , 1.0 MgCl ₂ , 10.0 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 10.0 glucose, pH adjusted to 7.35 with NaOH. The pipette solution contained (in mM): 75.0 CsCl, 64.0 Cs-gluconate, 1.0 MgCl ₂ , 10.0 HEPES, 3.0 Na ₂ ATP, pH adjusted to pH 7.3 with CsOH. Whole-cell currents were recorded using Axopatch 200B coupled to a 1322A digitizer. The patch pipette had a resistance of 3-5 MΩ. All recordings were performed at room temperature. Criteria for quantifying spontaneous transient inward currents (STICs) included currents with anamplitude twice the baseline as detected with pCLAMP10 and analysis by Origin 8 software (OriginLab Corporation, Northampton, MA). A voltage ramp from −60V to 40V was performed and STICs were recorded to generate a voltage-current relationship. In parallel studies, the impact of selective ANO 1/2 antagonists on STICs were analyzed. Briefly, following recording of spontaneous STICs, 100 μM benzbromarone, a powerful and selective ANO 1/2 antagonist was applied and STICs were measured. In addition, the effect of benzbromarone on G protein-coupled receptors and ryanodine receptor mediated activated STICs was also studied. All drugs used were reconstituted from stock solutions to final working concentration on the day of the experiment. Optimal concentrations were derived from preliminary dose response experiments. Benzbromarone was dissolved in dimethyl sulfoxide, the final concentration of which was less than 0.05%.

MUSM organ bath contractility studies

USM tissue was harvested from euthanized C57-Bl6 mice. The 5 mM circular segments of intact USM were cut from the uterine horn above the bifurcation. Segments were cleaned from mesenteric attachments. Ringed segments were placed in 8 mL organ baths (DMT Tissue Organ Bath 720MO; Danish Myo Technology, Aarhus, Denmark) in a modified Krebs−Henseleit buffer (concentration in mM: NaCl 115.0, KCl 2.5, CaCl ₂ 1.9, MgSO ₄ 2.5, NaHCO ₃ 25.0, NaH ₂ PO ₄ 1.4, D-glucose 5.6). Muscle force was digitally recorded during all experiments using BioPac hardware and AcqKnowledge 3.7.3 software (Biopac Systems, Inc., Goleta, CA). Rings were equilibrated to 1.0 g of isometric tension for 1 hour with Krebs–Henseleit buffer, based on preliminary experiments performed to determine the optimal starling forces for organ bath contractility (data not shown). Buffer was warmed to 37°C, replaced every 20 minutes, and continuously bubbled with 95% O ₂ /5% CO ₂ . Following buffer exchanges and resetting of tension to 1.0 g, rings were allowed to contract spontaneously. After reaching steady state for contractile frequency and amplitude, rings were then randomly treated with vehicle, tannic acid 100 μM (an ANO1/2 selective antagonist), or niflumic acid 100 μM (a ubiquitous chloride channel blocker was used as a positive control). The 100 μM dose for tannic acid was chosen based on preliminary dose response organ bath studies that yielded an IC ₅₀ of 96 μM (data not shown). Following addition of these drugs, muscle force was analyzed over the next 30 minutes examining changes in mean amplitude of force, and frequency compared with both time matched and vehicle treated controls. The pH was measured and maintained at 7.4 throughout the experiment in both the control and treatment groups with no significant change noted after addition of tannic or niflumic acid. Drugs were made from stock solutions and diluted to final desired concentrations.

Calcium flux studies

All calcium studies were performed using the ratiometric fluorescent calcium indicator Fura-2 (Calbiochem, Billerica, MA) as previously described. MUSM cells were grown to 100% confluence in 96 well black-walled clear-bottom plates between passages 4-10. Cells were washed with modified Hanks’ balanced salt solution (HBSS; concentration in mM: NaCl 137.9, KCl 5.3, CaCl ₂ 2.0, MgSO ₄ 1.0, HEPES 2.4, glucose 5.5, pH to 7.4). The cells were then loaded with 100 μL of 5 μM Fura-2 AM in a humidified 37°C incubator (95% air/5% CO ₂ ) for 30 minutes. Cells were washed again with HBSS and then incubated in HBSS for 20 minutes to allow for deesterification of the indicator. Cells received pretreatments with either an ANO1/2 specific antagonist (benzbromarone or tannic acid), a distinct ubiquitous chloride channel antagonist (5-Nitro-2-3-phenylpropylamino benzoic acid [NPPB]) was used as a positive control, or vehicle as a negative control for 10 minutes before study. After this incubation, cells were exposed to either oxytocin or bradykinin to induce GPCR mediated calcium release. The experiment was performed bradykinin as a Gq coupled agonist to confirm that the chloride channel antagonists’ effect was not limited to action via the oxytocin receptor. Fluorescence was measured in real time at 37°C using a Flex Station 3 (Molecular Devices, LLC, Sunnyvale, CA) using excitation wavelengths of 340 and 380, an emission wavelength of 510, and a cutoff filter of 495. Fluorescence values were reported as F/Fo according to the calculation:

Stay updated, free articles. Join our Telegram channel

Join